Abstract Cumin (Cuminum cyminum L.) is one of the most important medicinal and spicy herbs in the world. There is no report on the relationships between physio-biochemical characters, embryo regeneration and limonene synthase gene expression in cumin. This experiment was conducted to study physio-biochemical characters, embryo regeneration, limonene synthase gene expression, and their relationship in superior Iranian cumin landraces (Northern Khorasan, Kerman, Semnan, and Golestan) during two years. Results showed that Kerman landrace was superior in terms of embryo regeneration rate (89.33%), limonene synthase gene expression (17.2 fold), proline (3.9 mg/g FW), protein (3.35 mg/g FW), soluble sugars (9.8 mg/g FW), carotenoids (76 mg/g FW), chlorophyll b (16.17 mg/g FW), and monoterpene hydrocarbons (34.7%) contents. Embryo regeneration rate was negatively correlated with essential oil (r = −0.78*) and it was positively correlated with limonene synthase gene expression (r = 0.94**) and physio-biochemical characters including peroxidase activity (r = 0.95**) and the contents of proline (r = 0.82*), protein (r = 0.88*), soluble sugars (r = 0.99**), carotenoids (r = 0.97**), chlorophyll a (r = 0.95**), chlorophyll b (r = 0.84*), chlorophyll a + b (r = 0.91**), limonene (r = 0.94**), and monoterpene hydrocarbons (r = 0.93**). There was a positive and significant correlation between embryo regeneration rate and altitude (r = 0.75*). So it is concluded that embryo regeneration of cumin landraces are dependent of changes in altitude of their origins. The positive regression and correlation coefficients of soluble sugars with embryo regeneration rate indicate that this character is a suitable selective index for improving embryo regeneration rate. Considering the narrow-sense heritability of soluble sugars, this character can be used in breeding of embryo regeneration.